Combining Magnetically Isolated CD45 Cells with Serum Maintains Intact Drug Responsiveness for ELISpot Analysis in Clinical Trials

Overview

Journal Immunohorizons

Date 2025 Jan 30

PMID 39882960

Authors

Chris Mavrangelos

Asiri Wijenayaka

Kurt J Sales

Patrick A Hughes

Affiliations

Soon will be listed here.

Abstract

Enzyme-linked immunosorbent spot analysis is frequently used to investigate immune responsiveness during clinical trials. However, ELISpot classically utilizes peripheral blood mononuclear cell isolates from whole blood, requiring relatively high blood draw volumes and removing both granulocytes and bound drug. Here, we describe a novel protocol whereby CD45 cells are magnetically isolated from human whole blood and co-incubated with serum isolated from the same subject. Infliximab is a well characterized anti-tumor necrosis factor α (TNF-α) antibody in clinical use since the late 1990s. We demonstrated that TNF-α inhibition by infliximab in spiked whole blood is lost on peripheral blood mononuclear cell isolation but remains in serum, and that combining serum from infliximab spiked whole blood with magnetically isolated CD45 immune cells inhibited PMA/ionomycin-stimulated TNF-α secretion. This novel protocol has important implications for enzyme-linked immunosorbent spot analysis in clinical trials in which blood volume is limited, and keeping drug responses intact provides critical information.

References

Schnatbaum K, Holenya P, Pfeil S, Drosch M, Eckey M, Reimer U . An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays. Methods Mol Biol. 2024; 2768:29-50. DOI: 10.1007/978-1-0716-3690-9_3. View

Sedgwick J, Holt P . Kinetics and distribution of antigen-specific IgE-secreting cells during the primary antibody response in the rat. J Exp Med. 1983; 157(6):2178-83. PMC: 2187046. DOI: 10.1084/jem.157.6.2178. View

Hope C, Huynh D, Wong Y, Oakey H, Perkins G, Nguyen T . Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples. Int J Mol Sci. 2021; 22(17). PMC: 8431655. DOI: 10.3390/ijms22179129. View

Soares A, Govender L, Hughes J, Mavakla W, de Kock M, Barnard C . Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation. J Immunol Methods. 2010; 362(1-2):43-50. PMC: 2989440. DOI: 10.1016/j.jim.2010.08.007. View

Janetzki S, Schaed S, Blachere N, Ben-Porat L, Houghton A, Panageas K . Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods. 2004; 291(1-2):175-83. DOI: 10.1016/j.jim.2004.06.008. View

Islam R, Vance J, Poirier M, Zimmer J, Khadang A, Williams D . Recommendations on ELISpot assay validation by the GCC. Bioanalysis. 2022; 14(4):187-193. DOI: 10.4155/bio-2022-0010. View

Slota M, Lim J, Dang Y, Disis M . ELISpot for measuring human immune responses to vaccines. Expert Rev Vaccines. 2011; 10(3):299-306. PMC: 3360522. DOI: 10.1586/erv.10.169. View

Tzeng S . The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity. J Vis Exp. 2017; (118). PMC: 5226391. DOI: 10.3791/54582. View

Janetzki S, Panageas K, Ben-Porat L, Boyer J, Britten C, Clay T . Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother. 2007; 57(3):303-15. PMC: 2150634. DOI: 10.1007/s00262-007-0380-6. View

10.

Cornillie F, Shealy D, DHaens G, Geboes K, Van Assche G, Ceuppens J . Infliximab induces potent anti-inflammatory and local immunomodulatory activity but no systemic immune suppression in patients with Crohn's disease. Aliment Pharmacol Ther. 2001; 15(4):463-73. DOI: 10.1046/j.1365-2036.2001.00956.x. View

11.

Hughes P, Moretta M, Lim A, Grasby D, Bird D, Brierley S . Immune derived opioidergic inhibition of viscerosensory afferents is decreased in Irritable Bowel Syndrome patients. Brain Behav Immun. 2014; 42:191-203. DOI: 10.1016/j.bbi.2014.07.001. View

12.

Hughes P, Harrington A, Castro J, Liebregts T, Adam B, Grasby D . Sensory neuro-immune interactions differ between irritable bowel syndrome subtypes. Gut. 2012; 62(10):1456-65. DOI: 10.1136/gutjnl-2011-301856. View

13.

Grutzkau A, Radbruch A . Small but mighty: how the MACS-technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years. Cytometry A. 2010; 77(7):643-7. DOI: 10.1002/cyto.a.20918. View

14.

Jahnmatz M, Kesa G, Netterlid E, Buisman A, Thorstensson R, Ahlborg N . Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses. J Immunol Methods. 2013; 391(1-2):50-9. DOI: 10.1016/j.jim.2013.02.009. View

15.

Miwa H, Dimatteo R, de Rutte J, Ghosh R, Di Carlo D . Single-cell sorting based on secreted products for functionally defined cell therapies. Microsyst Nanoeng. 2022; 8:84. PMC: 9303846. DOI: 10.1038/s41378-022-00422-x. View

16.

Czerkinsky C, Svennerholm A . Ganglioside GM1 enzyme-linked immunospot assay for simple identification of heat-labile enterotoxin-producing Escherichia coli. J Clin Microbiol. 1983; 17(6):965-9. PMC: 272784. DOI: 10.1128/jcm.17.6.965-969.1983. View

17.

Lehmann P, Zhang W . Unique strengths of ELISPOT for T cell diagnostics. Methods Mol Biol. 2011; 792:3-23. DOI: 10.1007/978-1-61779-325-7_1. View

18.

Costantini A, Mancini S, Giuliodoro S, Butini L, Regnery C, Silvestri G . Effects of cryopreservation on lymphocyte immunophenotype and function. J Immunol Methods. 2003; 278(1-2):145-55. DOI: 10.1016/s0022-1759(03)00202-3. View

19.

Zola H, Swart B, Nicholson I, Aasted B, Bensussan A, Boumsell L . CD molecules 2005: human cell differentiation molecules. Blood. 2005; 106(9):3123-6. DOI: 10.1182/blood-2005-03-1338. View

20.

Sedgwick J, Holt P . A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J Immunol Methods. 1983; 57(1-3):301-9. DOI: 10.1016/0022-1759(83)90091-1. View